Experiments were initiated on two mouse cell lines, a parental line carrying one copy of SV40 DNA per diploid equivalent of cell DNA and a subline, obtained by immunoselection, carrying about 0.5 copies of SV40 DNA per diploid equivalent of cell DNA. Cloned derivatives of both lines were stable. The parental line synthesized early SV40 RNA and expressed early SV40 antigens. The subline had lost early SV40 gene sequences and produced no detectable virus specific RNA nor virus antigens. To explore possible mechanisms for the SV40 gene loss restriction enzyme-cleaved fragments of DNA were obtained from each line and were subjected to gel electrophoresis, denatured and transferred to nitrocellulose paper by the Southern technique. Integrated SV40 DNA fragments were localized by hybridization to a labeled SV40 DNA probe. Preliminary results show differences in SV40 DNA distribution between the two cells DNAs, which will eventually provide an insight into the mechanism by which SV40 DNA sequences were lost from the T-antigen negative clone.